Plant-derived carotenoids are converted in humans to vitamin A. Vitamin A can not be synthesized de novo and it is essential for growth and development. Deficiencies manifest as xerophthalmia, blindness, increased mortality due to increased severity of childhood diseases and increased maternal transmission of viruses such as HIV. An approach to alleviating vitamin A deficiency worldwide is to improve levels of provitamin A carotenoids in food staples such as corn, wheat, and rice by metabolic engineering. Preliminary success with metabolic engineering of the pathway in plants points to the potential of this approach. Unexpected products in such transgenic plants, however, suggest that the technology is limited by current deficiencies in understanding of endogenous gene expression. Rational metabolic engineering strategies must take into account the regulation of the endogenous pathway which is not yet completely understood. We propose that the relative accumulation of provitamin A carotenoids is mediated by control of transcript levels for the biosynthetic enzymes; and that appropriate modification (enhancement or repression) of transcript levels in maize endosperm can lead to increased levels of provitamin A carotenoids relative to other nonprovitamin A carotenoids. NIH SCORE funding has allowed us to begin to develop tools and research which are most immediately applicable to two of the most important food crops worldwide- corn and rice. We discovered that several pathway enzymes are encoded by small gene families, which raises the question: what is the contribution of gene family members to carotenoid biosynthesis and accumulation in different tissues, developmental stages, and plastid membrane localization. We will continue our NIH-SCORE supported work to conduct a comprehensive investigation of expression of endogenous genes which encode enzymes of the carotenoid pathway and the related isoprenoid biosynthetic pathway enzymes needed to provide substrates for the carotenoid pathway. We will isolate remaining genes; and characterize gene families in terms of gene structure, transcript accumulation in developing endosperm and other tissues, and test function and localization of gene products. We will address the following questions: 1) Do gene family members differ in tissue and/or temporal specificity of expression? 2) Which gene family members encode functional proteins? 3) Are gene products of family members targeted differentially to plastid envelope vs. thylakoid membranes?